Malignant melanoma cells acquire resistance to DNA interstrand cross-linking chemotherapeutics by p53-triggered upregulation of DDB2/XPC-mediated DNA repair.

Malignant melanoma is a cancer characterized by high chemoresistance although p53 is rarely mutated. Here, we show that p53 wild-type melanoma cells acquire resistance to cell death induced by fotemustine (FM), which is a representative of alkylating DNA interstrand cross-linking agents used in melanoma therapy. We show that drug-induced resistance is a result of p53-dependent upregulation of the nucleotide excision repair (NER) genes xeroderma pigmentosum complementation group C (XPC) and damaged DNA-binding protein 2 (DDB2), which stimulate the repair of DNA interstrand cross-links (ICLs) arising from O(6)-chloroethylguanine. Consequently, TP53 mutated cells are unable to repair ICLs, leading to prolonged ATM, ATR and checkpoint kinase 1 (CHK1) activation, and finally apoptosis. The roles of p53 and NER in ICL-triggered cell death were confirmed by knockdown of p53 and XPC. Upregulation of XPC and DDB2 in p53wt cells following a single drug treatment is a robust and sustained response that lasts for up to 1 week. Pretreatment with an inducing dose followed by a high and toxic dose of FM provoked an adaptive response as the killing outcome of the challenge dose was reduced. Upregulation of XPC and DDB2 was also observed in a melanoma mouse xenograft model following systemic administration of FM. Additionally, XPC and DDB2 induction occurred upon treatment with other cross-linking anticancer drugs, such as cisplatin and mafosfamide, indicating it is a general response of cancer cells to this group of chemotherapeutics. Collectively, the data indicate that p53-dependent upregulation of XPC and DDB2 is a key mechanism upon genotoxic stress, whereby melanoma cells acquire resistance towards DNA cross-linking agents. To our knowledge, this is the first demonstration of upregulation of NER following a single dose of a DNA interstrand cross-linker, which is a robust and long-lasting effect that impacts the killing response of cancer cells to subsequent treatments.

Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53.

Malignant melanomas are highly resistant to chemotherapy. First-line chemotherapeutics used in melanoma therapy are the methylating agents dacarbazine (DTIC) and temozolomide (TMZ) and the chloroethylating agents BCNU and fotemustine. Here, we determined the mode of cell death in 11 melanoma cell lines upon exposure to TMZ and fotemustine. We show for the first time that TMZ induces apoptosis in melanoma cells, using therapeutic doses. For both TMZ and fotemustine apoptosis is the dominant mode of cell death. The contribution of necrosis to total cell death varied between 10 and 40%. The O(6)-methylguanine-DNA methyltransferase (MGMT) activity in the cell lines was between 0 and 1100 fmol mg(-1) protein, and there was a correlation between MGMT activity and the level of resistance to TMZ and fotemustine. MGMT inactivation by O(6)-benzylguanine sensitized all melanoma cell lines expressing MGMT to TMZ and fotemustine-induced apoptosis, and MGMT transfection attenuated the apoptotic response. This supports that O(6)-alkylguanines are critical lesions involved in the initiation of programmed melanoma cell death. One of the cell lines (MZ7), derived from a patient subjected to DTIC therapy, exhibited a high level of resistance to TMZ without expressing MGMT. This was related to an impaired expression of MSH2 and MSH6. The cells were not cross-resistant to fotemustine. Although these data indicate that methylating drug resistance of melanoma cells can be acquired by down-regulation of mismatch repair, a correlation between MSH2 and MSH6 expression in the different lines and TMZ sensitivity was not found. Apoptosis in melanoma cells induced by TMZ and fotemustine was accompanied by double-strand break (DSB) formation (as determined by H2AX phosphorylation) and caspase-3 and -7 activation as well as PARP cleavage. For TMZ, DSBs correlated significantly with the apoptotic response, whereas for fotemustine a correlation was not found. Melanoma lines expressing p53 wild-type were more resistant to TMZ and fotemustine than p53 mutant melanoma lines, which is in marked contrast to previous data reported for glioma cells treated with TMZ. Overall, the findings are in line with the model that in melanoma cells TMZ-induced O(6)-methylguanine triggers the apoptotic (and necrotic) pathway through DSBs, whereas for chloroethylating agents apoptosis is triggered in a more complex manner.

Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine.

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O(6)-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O(6)-benzylguanine showed that, in gliomas, the apoptotic signal originates from O(6)-methylguanine (O(6)MeG) and that repair of O(6)MeG by MGMT prevents apoptosis. We further demonstrate that O(6)MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O(6)MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O(6)MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by gammaH2AX formation. Glioma cells mutated in DNA-PK(cs), which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.